A real-world study assessing the impact of retinal fluid on visual acuity outcomes in patients with neovascular age-related macular degeneration in Korea

To evaluate the real-world treatment outcomes in patients with neovascular age-related macular degeneration (nAMD) in Korea, focusing on retinal fluid resolution. This multi-institutional retrospective chart review study, analyzed medical records of patients with nAMD (age ≥ 50 years) who received their first anti-vascular endothelial growth factor (VEGF) treatment in ophthalmology clinics across South Korea between January 2017 and March 2019. The primary endpoint was the proportion of patients with retinal fluid after 12 months of anti-VEGF treatment. The association between fluid-free period and VA gains was also evaluated. A total of 600 patients were enrolled. At baseline, 97.16% of patients had retinal fluid; after 12 months of anti-VEGF treatment, 58.10% of patients had persistent retinal fluid. VA improvements were relatively better in patients with absence of retinal fluid compared with presence of retinal fluid (+ 12.29 letters vs. + 6.45 letters at month 12; P < .0001). Longer duration of absence of retinal fluid over first 12 months correlated with better VA gains at month 12 (P < .01). More than half of the study patients with nAMD had retinal fluid even after 12 months of treatment with their current anti-VEGF. Presence of retinal fluid was associated with relatively worse VA outcomes.

Visual acuity changes by presence of retinal fluid at each time point. VA gains from baseline were significantly greater in patients with absence of retinal fluid versus those with presence of retinal fluid, with a P value of < 0.05 at months 3, 6, 9, 12, 15, and 21. At months 18 and 24, though VA gains were higher in the absence of retinal fluid compared to presence of fluid, the difference in between the two groups was not significant (P value > 0.05). At month 12, the mean difference in VA gain between patients with absence of retinal fluid versus those with presence of retinal fluid was 5.84 letters. Similarly, at any given time point, VA gains were relatively better in patients with absence of IRF, SRF, and sub-RPE fluid. (Fig. 4) The mean VA at each time point is shown in Fig. S1.
Visual acuity changes by presence of retinal fluid at month 3. We assessed the VA outcome at all time points based on the presence/absence of retinal fluid and the types of retinal fluid only at month 3 (after the loading dose for anti-VEGF therapy) (Fig. 5). VA gains were relatively better in patients who achieved absence of retinal fluid at month 3 versus those with presence of retinal fluid, with a P value of < 0.05 at months 3 and 6. VA gains were numerically greater in patients with absence of SRF and sub-RPE fluid at month 3, from month 3 to approximately month 24. However, the difference was not enough to attain statistical significance (P Table 1. Patient demographics and characteristics (Cohort 1). CNV, choroidal neovascularization; DA, disc area; n, number of patients; N, total number of patients; PCV, polypoidal choroidal vasculopathy; RAP, retinal angiomatous proliferation; SD, standard deviation. *Index anti-VEGF, anti-VEGF injection taken during the first/baseline visit. Visual acuity change by quartile time with absence of retinal fluid. After categorizing patients into quartiles based on time of absence with retinal fluid, patients with a longer duration of absence of retinal fluid from 1 to 12 months showed higher VA gains. There were substantial differences in VA gains at month 12 for the Q1-Q2 subgroup compared with Q3 (P = 0.0038) and Q4 subgroups (P = 0.0004) (Fig. 6a). Similarly, when patients were divided into quartiles based on time of absence of retinal fluid from 1 to 24 months, VA gains at month 24 were also relatively higher in the group with a longer duration of absence of retinal fluid. However, the P values for the differences between the groups were > 0.05 (Fig. 6b).

Discussion
To the best of our knowledge, the PROOF study is the first multicenter study to assess the efficacy of anti-VEGFs in resolving retinal fluid in nAMD in the real-world setting in Korea. This study found that despite their current anti-VEGF treatment, a high proportion of patients with nAMD continued to have retinal fluid at year 1 as well as year 2 of treatment. Further, patients who achieved early fluid resolution showed a trend towards better VA gains. Disease activity in nAMD has been defined based on 3 parameters: (1) a loss of ≥ 5 letters in VA, (2) evidence of new hemorrhage, and (3) the presence of retinal fluid, including IRF and SRF 17 . Clinical practice guidelines from the American Academy of Ophthalmology 14 and the European Society of Retina Specialists 13 recommend retreatment with anti-VEGFs if retinal fluid is detected on OCT as it is an indication of active disease. A nAMD study from United Kingdom including 1190 eyes showed that presence of IRF and SRF has greater influence on real-world anti-VEGF retreatment decisions compared to the presence of pigment epithelial detachment or vision loss 18 .
In the present study, the reduction in the proportion of patients with retinal fluid was most noticeable by 3 months (after the loading dose), although only 54.55% of patients achieved fluid-free status. However, > 50% of the patients treated with anti-VEGFs had retinal fluid at the end of both year 1 and year 2 of the treatment. One possible reason for the high proportion of patients without resolution of retinal fluid in this study was the use of PRN regimen in nearly half of the patients. However, a subset of patients may have been either undertreated, or they did not respond adequately to the current anti-VEGF treatments and/or could have required a more intensive treatment. In the current study, anti-VEGFs received by patients with nAMD in Korea included ranibizumab, aflibercept, and off-label use of bevacizumab. A treatment that could more effectively provide better disease control without increasing the treatment burden is an unmet need for nAMD management in Korea.
There are several emerging treatments for nAMD that are under investigation, including longer-acting anti-VEGF agents, sustained systems, topical treatments, gene therapy, and molecules targeting novel angiogenesis checkpoints 19,20 . Notably, there are novel agents that can extend treatment intervals up to 12 to 16 weeks [21][22][23] . The most recent anti-VEGF therapy for nAMD includes brolucizumab, a single-chain antibody fragment inhibitor of all isoforms of VEGF-A 21 . In phase 3 studies, brolucizumab 6 mg demonstrated a better disease control including superior fluid resolution, with > 50% patients treated on a q12w interval after loading up to week 48 versus aflibercept 2 mg dosed q8w 21,22 . However, higher incidence of intraocular inflammation (IOI) was reported in patients treated with brolucizumab (4.7%) compared to aflibercept (< 1%) 21,22 . Following post-marketing reports, a rare safety signal of adverse event of retinal vasculitis (RV) and/ or retinal vascular occlusion (RO) which may result in severe vision loss was identified with brolucizumab.These events typically occured in the presence of IOI [24][25][26] . The VA outcome in this study mirrored the trend observed for retinal fluid status, with the maximum improvement by 3 months after anti-VEGF treatment. In this study, the mean VA gains at month 3 were maintained through 12 months and showed a trend of decline towards 24 months. The VA gain reported in the present study at 12 months (mean + 8.91 letters with 5.54 injections) was relatively higher than in other realworld studies. A systematic review and meta-analysis that assessed the real-world effectiveness of ranibizumab and aflibercept in treatment-naive nAMD patients showed that the VA gain (ETDRS) and injection numbers at 12 months were + 4.24 letters and 5.88 injections with ranibizumab treatment, and + 5.30 letters and 7.10 injections with aflibercept treatment 28 . In the global LUMINOUS study, treatment-naive nAMD patients treated with ranibizumab for 1 year showed a mean VA gain of 3.1 letters with 5.0 injections 29 . A real-world study from US that included 2213 nAMD patients showed that patients treated with anti-VEGFs (ranibizumab, aflibercept, or bevacizumab) for 2 years had a mean change in VA of + 3.1 letters from baseline with 12.1 injections 30 .
This study demonstrates that the retinal fluid status at each time point influenced the VA outcome, whereby patients with absence of retinal fluid had better VA gains than patients with presence of retinal fluid. The same trend was observed for the individual retinal fluid components, including IRF, SRF, and sub-RPE fluid. In some previous studies the presence of SRF was not found to be associated with poor visual outcome 31,32 . Similarly, Core et al., reported that the long-term persisting SRF did not have an impact on VA outcomes through 2 years of follow-up in nAMD patients 33 . However, several recent studies demonstrated contrary results 18,34 . Post hoc analysis of the FLUID study suggested that IRF in the central 1 mm and SRF in the 1 to 6 mm of macular area were negatively associated with best-corrected VA 34 .
Another study with 321 eyes with nAMD showed that absence of IRF or SRF at ≥ 2 clinic visits resulted in a gain of 5 ETDRS letters from baseline, compared with 2 letters gained in eyes with < 2 clinic visits with absence of IRF or SRF 18 . The question of whether SRF can be tolerated is still being debated. In the present study, patients with absence of SRF had overall better VA gain compared with those who had SRF, especially at 12 months after treatment, with 4.52 letters difference in VA gain between the 2 groups. This result may indicate potential negative impact of SRF on visual outcome, implying the need for appropriate SRF control.
We further assessed if the fluid status at month 3 affects the VA outcome at subsequent time points. Compared to patients with presence of retinal fluid, patients with absence of retinal fluid at month 3 achieved slightly better VA gains (~ 1-2 letters) from 3 to 21 months; analyses of SRF and sub-RPE fluid followed a similar pattern.   . S1 and S2). The ceiling effect, whereby patients with higher baseline VA have limited potential to gain more letters, while those with lower baseline VA have little possibility for further loss of vision, may have caused a relatively lower VA gain in the patient subgroup with absence of IRF and vice versa. This finding is in agreement with previous studies, which have reported that the presence of IRF at baseline and during treatment is detrimental to vision 35,36 . It is also in line with the findings from a retrospective 1-year follow-up study of anti-VEGF-treated nAMD eyes showing that presence of IRF was associated with a lower VA at baseline 37 .Patients with presence of IRF at baseline represent a population with more advanced progression of nAMD, highlighting the need to treat IRF aggressively until maximum resolution is achieved 38 . Recently, Core et al. reported the presence of IRF in ≥ 80% of visits through year 2 was closely associated with worse long-term VA and scar development in nAMD patients, confirming the negative impact of persisting IRF on visual prognosis 39 . Our results confirmed that the presence of IRF, SRF, and sub-RPE fluid is associated with a worse VA outcome and suggest that all retinal fluids are pathological. In general, the presence of sub-RPE fluid is not considered to be closely associated with visual prognosis 40 . But few studies have suggested that the sub-RPE fluid can be associated with the prognosis in some circumstances 41,42 .
In addition, the incidence of sub-RPE fluid has not been studied in a large Korean cohort. For these reasons, the incidence and clinical significance of sub-RPE fluid were analyzed in this study. We further assessed the effect of time of absence of retinal fluid on VA outcomes. Patients who were fluidfree for a longer time had relatively better VA gains at both 12 and 24 months, as compared to patients with the shortest time with absence of retinal fluid. This result highlights the importance of long-term fluid control.
In the current study, a similar proportion of patients were treated using the PRN or T&E treatment regimen, with a small proportion of patients adopting an approach of a combination of both regimens. The number of injections and visits for treatment-naive patients with nAMD was 5.54 and 9.51, respectively, in year 1, and 3.48 and 7.10, respectively, in year 2. This was consistent with the real-world ranibizumab treatment pattern for the Korean patient population with nAMD in the LUMINOUS study, in which treatment-naive patients received 5.2 injections and 9.2 monitoring visits by year 1 43 . The number of injections and visits was relatively higher than that in real-world studies conducted in other regions [44][45][46] but was relatively lower than that in a study conducted in the US 47 . By 12 months, the patients achieved a VA improvement of + 8.1 letters and CST reduction of 106.90 µm. The results were comparatively better than those from other real-world studies for ranibizumab and aflibercept 28,30,48 .
Overall, the findings of this study are clinically meaningful as they reflect the actual clinical settings in South Korea and provide insights to fluid management strategies. The demographics and clinical characteristics of the study population were consistent with that reported in previous real-world studies from Korea 6,43 . Further, the study included a large sample population of 600 nAMD patients, adding strength to the findings.
Limitations include the retrospective nature of the study. The study cohort was heterogeneous compared to clinical trials. In addition, patients who received ranibizumab, aflibercept, or bevacizumab were pooled in the same cohort, which also included patients who had received two or more different anti-VEGF agents during treatment. Since efficacy might differ amidst the different anti-VEGF agents used, this serves as a major limitation of the study. Decisions regarding treatment regimens and schedule were made based on the physicians' discretion (PRN or T&E and continuation/discontinuation/switch of treatment). Assessment variables, such as collection and interpretation of retinal images, may also have differed based on the institutions' practice. Furthermore, the present study evaluated the influence of each fluid compartment on visual outcome separately. For this reason, we could not evaluate the influence of co-existing two or more fluid compartments. In addition, the difference in the impact of different fluid locations (foveal vs extrafoveal) was not evaluated. Lastly, since the current study focused on real-world anti-VEGF treatment patterns, we did not include safety data, which are well-known and reported in the prior literature.
In conclusion, our study found that a high proportion of Korean patients with nAMD on currently available anti-VEGF therapies had reported presence of retinal fluid at year 1 and year 2. Patients who achieved early fluid-free status following anti-VEGF treatment and those who had a longer time of absence of retinal fluid show a trend toward better visual outcomes. New and emerging anti-VEGF therapies that can provide sustained disease control with longer duration of action could lead to improved outcomes and address the high treatment burden for patients requiring more aggressive treatment, thus potentially fulfilling the unmet needs of patients with nAMD in Korea.

Methods
Study design and treatment. The PROOF study was a retrospective chart review of patients with nAMD who received their first anti-VEGF treatment between January 2017 and March 2019 in 10 ophthalmology clinics across South Korea. The primary objective of the study was to evaluate the presence of retinal fluid in patients with nAMD after 1 year of anti-VEGF treatment. The study was approved by institutional review board/independent ethics committee for each participating center.
The study period was between July 1, 2016 and March 31, 2020, which allowed for a 6-month pre-index period and an at least 12-month follow-up period. The index/baseline date was defined as the date of the first anti-VEGF injection in patients' eyes. Patients were administered anti-VEGF treatment, which included ranibizumab, bevacizumab, or aflibercept; the choice of treatment and the decision to discontinue treatment were at the discretion of the prescribing investigator and the patient. www.nature.com/scientificreports/ indication, or clinical findings on or within 6 months prior to the index date; (3) were ≥ 50 years of age at baseline; (4) had retinal fluid status on OCT captured at baseline and at 1 year of follow-up; and (5) had undergone standard examination procedures, including retinal imaging (eg, fundus photography and OCT) at baseline and follow-up, were included. Patients were excluded if they had received any other anti-VEGF and/or photodynamic therapy in the study eye prior to baseline, had concurrent progressive retinal diseases requiring treatment to prevent vision loss (eg, diabetic macular edema, retinal vein occlusion), were receiving any other treatments such as intravitreal steroids or cataract surgery, or participating in any other clinical trial during the study period. Based on fluorescein angiography (FA) results, the location of choroidal neovascularization (CNV) was categorized into three: subfoveal (involving the foveal center), juxtafoveal (1-199 µm from the foveal center), and extrafoveal (≥ 200 µm from the foveal center). The CNV lesion composition was also classified based on FA results, according to the previously suggested criteria: predominantly classic (the area of classic CNV was at least 50% of the area of the entire lesion), minimally classic (the area of classic CNV was < 50% of the area of the entire lesion, and occult (lesions without classic CNV component) 49 . Secondary data captured in the medical records were extracted retrospectively as the source of the data. An electronic data capture (EDC) method, provided by LSK Global Pharma Services Co. Ltd., Korea, was used in this study; anonymized patient data from the study site were entered directly into the database by the investigator or investigator's designee via a web browser to establish the patients' electronic case report form (eCRF). The EDC system verified the integrity of the project progression by conducting system validation.

Endpoints.
Statistical analyses. The primary analysis was performed in patients who met the inclusion criteria of the study and had a minimum follow-up for a period of 12 months (cohort 1, n = 600). The longer-term outcomes up to 24 months were additionally evaluated in a subset of patients from cohort 1 who had a minimum follow-up of 24 months (cohort 2, n = 453).
For the primary endpoint of the proportion of patients with retinal fluid during the first year of treatment, precision was calculated based on the following assumptions that at least 70% of the patients have retinal fluid and patients would have at least 1 year of follow-up (N > 600). With a sample size of 600, the 2-sided 95% confidence interval (CI) for π = 0.7 was estimated to range between 66 and 74%. Under the worst case scenario of π = 0.5, the 2-sided 95% CI would range between 46 and 54%.
This study was exploratory in nature, and thus, no comparative analysis was planned. All patients who received any anti-VEGFs for nAMD were pooled in the same cohort, and there was no censoring at the time of switching to another anti-VEGF. In patients who received bilateral treatment, only the first treated eye was analyzed. Descriptive statistics (number of observations, proportions, means and standard deviations [SDs], median and minimum and maximum values, and 2-sided 95% CIs of the mean) were tabulated for the demographic and clinical characteristics and all outcome variables unless otherwise stated. Two-sided 95% CIs were derived for all proportions. No data imputation was performed.
For VA analysis, either the naked eye vision or corrected vision was used for each patient. VA was analyzed in subgroups stratified by (1) absence/presence of retinal fluids at each time point: VA at month N was stratified by absence/presence of fluids at month N; (2) absence/presence of retinal fluids at month 3: VA at all time points was stratified by absence/presence of fluids at month 3; (3) time with absence of fluid over 1 to 12 months (cohort 1) and 1 to 24 months (cohort 2): time was calculated as the sum of fluid-absence duration (time from the absence of fluid to a day before subsequent presence from day 1 to 360 and day 1 to 720 in days). Patients were divided into quartiles groups based on their time with absence of fluid. Quartile values for cohort 1: lower quartile = 0.0 day, median = 1.0 day, upper quartile = 139.5 days; quartile values for cohort 2: lower quartile = 1.0 day, median = 86.0 days, upper quartile = 288.0 days.
The Wilcoxon signed-rank test was used to assess the difference in VA change between patients with presence and absence of retinal fluid, and in patients with different quartiles of time with absence of retinal fluid. Two-sided P values were reported. All analyses were performed by LSK Global Pharma Services Co. Ltd., Korea, using Statistical Analysis System (SAS) version 9.4 software.
Ethical approval. This study was conducted in accordance with the Guidelines for Good Pharmacoepidemiology Practices issued by the International Society for Pharmacoepidemiology and the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines, and adhered to the ethical principles derived from the Declaration of Helsinki. Each center's institutional review board/independent ethics committee (IRB/IEC) reviewed and approved the study protocol.
Informed consent. Patients from Samsung Medical Center provided written informed consent, and the IRB/IEC of the other study sites approved the waiver of patient consent. All research adhered to the tenets of the Declaration of Helsinki. No animal subjects were included in this study.